The present invention is generally in the area of methods and treatments for central nervous system injury, and particularly the use of trophic factors for spinal cord regeneration.
Past early childhood, injury to the central nervous system (CNS) results in functional impairments that are largely irreversible. Within the brain or spinal cord, damage resulting from stroke, trauma, or other causes can result in life-long losses in cognitive, sensory and motor functions, and even maintenance of vital functions. Nerve cells that are lost are not replaced, and those that are spared are generally unable to regrow severed connections, although a limited amount of local synaptic reorganization can occur close to the site of injury. Functions that are lost are currently untreatable.
Regenerative failure in the CNS has been attributed to a number of factors, which include the presence of inhibitory molecules on the surface of glial cells that suppress axonal growth; absence of appropriate substrate molecules such as laminin to foster growth; and an absence of the appropriate trophic factors needed to activate programs of gene expression required for cell survival and differentiation.
By contrast, within the peripheral nervous system (PNS), injured nerve fibers can regrow over long distances, with eventual excellent recovery of function. Within the past 15 years, neuroscientists have come to realize that this is not a consequence of intrinsic differences between the nerve cells of the peripheral and central nervous system; remarkably, neurons of the CNS will extend their axons over great distances if given the opportunity to grow through a grafted segment of PNS (e.g., sciatic nerve). Therefore, neurons of the CNS retain a capacity to grow if given the right signals from the extracellular environment. Factors which contribute to the differing growth potentials of the CNS and PNS include partially characterized, growth-inhibiting molecules on the surface of the oligodendrocytes that surround nerve fibers in the CNS, but which are less abundant in the comparable cell population of the PNS (Schwann cells); molecules of the basal lamina and other surfaces that foster growth in the PNS but which are absent in the CNS (e.g., laminin); and trophic factors, soluble polypeptides which activate programs of gene expression that underlie cell survival and differentiation. Although such trophic factors are regarded as essential to maintaining the viability and differentiation of nerve cells, the particular ones that are responsible for inducing axonal regeneration in the CNS remain uncertain.
In contrast to man and other higher vertebrates, lower vertebrates are able to regenerate injured CNS pathways throughout life (Sperry, R. W. (1944), J. Neurophysiol., 7:57-69; Sperry, R. W. (1963), Proc. Nat. Acad. Sci. USA, 50:703-710). In the goldfish, 95% of retinal ganglion cells survive injury to the optic nerve (Meyer, et al., (1985), J. Comp. Neurol., 239:27-43) and go on to re-establish topographically organized, functional connections with cells of the optic tectum and other target areas within one to two months (reviewed in Grafstein, (1986), The retina as a regenerating organ, In R. Adler and B. D. Farber (Eds.), The Retina: A Model for Cell Biology Studies Part II, Academic Press, New York, 275-335; Jacobson, (1991), Development Neurobiology, third edition (Plenum Publishing Co., New York)). The cellular and molecular changes that accompany this process have been studied in depth. Retinal ganglion cells undergo extensive metabolic and morphological changes that include a dramatic enlargement of the nucleolus, a proliferation of free ribosomes, and an increase in cell diameter (Murray & Grafstein, (1969), Exp. Neurol., 23:544-560; Murray & Forman, 1971 (1971), Brain Res., 32:287-298). Massive increases are seen in the expression of genes encoding certain components of the cytoskeleton (Burrell, et al., (1978), J. Neurochem., 31:289-298; Heacock & Agranoff, (1982), Neurochem. Res., 7:771-788; Giulian, et al., (1980), J. Biol. Chem., 255:6494-6501; Quitschke & Schechter, (1983), Brain Res., 258:69-78; Glasgow, et al., (1994) EMBO J., 13:297-305; Glasgow, et al. (1992) Neuron, 9:373-381), cell surface adhesion molecules (Vielmetter, et al., (1991) J. Neurosci, 11:3581-3593; Bastmeyer, et al., (1990) Development, 108:299-311; Paschke, et al., (1992) J. Cell Biol., 117:863-875; Blaugrund, et al., 1990; Battisti, et al., (1992) J. Neurocytol., 21:557-73), and several proteins that become incorporated into the growing nerve terminal membrane, particularly GAP-43 (Benowitz, et al., (1981) J. Neurosci., 1:300-307; Heacock & Agranoff, (1982); Perrone-Bizzozero, et al., (1987), J. Neurochem., 48:644-652; Perry, et al., (1987), J. Neurosci., 7:792-806; LaBate & Skene, (1989), Neuron, 3:299-310; Wilmot, et al., (1993), J. Neurosci., 13:387-401). Some of the same changes are associated with the development and regeneration of the optic nerve in other species (Skene & Willard, (1981), J. Cell Biol., 89:86-95 J. Cell. Biol., 89:96-103; Moya, et al., (1988), J. Neurosci., 8:4445-4454; Doster, et al., (1991), Neuron, 6:635-647).
In general, the capacity of neurons to regenerate their axons after injury is strongly influenced by the surrounding non-neuronal elements (Aguayo, et al., (1991) Phil. Trans. Royal Soc. London, Series B, 331:337-343). In the case of the goldfish retinofugal pathway, the glial sheath cells of the optic nerve seem to provide an environment that is highly conducive to axonal outgrowth (Bastmeyer, et al., (1993) Glia, 8:1-11; Bastmeyer, et al., (1991) J. Neurosci, 11:626-640). In part, this may be attributed to the expression of particular cell surface and extracellular matrix proteins, including an L1-like cell adhesion molecule (Blaugrund, et al., (1990) Brain Res., 530:239-244; Bastmeyer, et al., (1993); Bastmeyer, et al. (1991); Vielmetter, et al., 1991; Battisti, et al., 1992), laminin (Hopkins, et al., (1985) J. Neurosci., 5:3030-3038), and chondroitin sulfate proteoglycans (Battisti, et al., 1992). At the same time, optic nerve glia of goldfish seem to express lower levels of growth-inhibiting proteins on their surfaces than mammalian CNS oligodendrocytes (Caroni & Schwab, (1988) J. Cell Biol., 106:1281-1288; Schwab & Caroni, (1988) J. Neurosci., 8:2381-2393; Bastmeyer, et al., 1991; Sivron, et al., (1994), Presence of growth inhibitors in fish optic nerve myelin: postinjury changes. J. Comp. Neurol., 343:237-246).
In addition to cell surface components, cells of the goldfish optic nerve secrete soluble factors that promote axonal outgrowth from goldfish retinal explants (Mizrachi, et al., (1986) J. Neurochem., 46:1675-1682), embryonic mammalian neurons (Finkelstein, et al., (1987) Brain Res., 413:267-274; Caday, et al., 1989), and the mature rabbit retina (Schwartz, et al., (1985) Science, 228:600-603). Among the proteins that are secreted by the glia and microphages of the optic nerve are apolipoprotein A (Harel, et al., (1989) J. Neurochem., 52:1218-1228), a plasminogen activator (Salles, et al., (1990) EMBO J., 9:2471-2477), interleukin-2 (Eitan, et al., 1992), a transglutaminase (Eitan and Schwartz, (1993) Science, 261:106-108), and platelet-derived growth factor (Eitan, et al., (1992) Proc. Natl. Acad. Sci. USA, 89:5442-5446).
Despite these findings, the factors responsible for initiating axonal outgrowth from retinal ganglion cells remain unknown. Studies directed towards this issue have generally been carried out either in vivo or have utilized retinal explants derived from animals in which regeneration had already been triggered in vivo by a conditioning lesion (Landreth and Agranoff (1976) Brain Res., 118:299-303; Landreth and Agranoff (1979) Brain Res., 161:39-53; Turner, et al., (1981) Brain Res., 204:283-294; Turner, et al. (1982) Dev. Brain Res., 4:59-66; Schwartz, et al., 1985; Yip & Grafstein, (1982) Brain Res., 238:329-339; Hopkins, et al., 1985; Lima, et al., (1989) Int. J. Devl. Neuroscience, 7:375-382). The fact that various agents tested fail to augment outgrowth unless the regenerative process had already begun in vivo suggests that the factors required to initiate regeneration may derive from a source that is absent in the explant cultures, e.g., the optic nerve glia, the circulatory system, or other brain tissue (Johnson and Turner, (1982) J. Neurosci. Res., 8:315-329). Trophic factors are generally reviewed in Developmental Neurobiology, M. Jacobson (Third Edition, Plenum Publ. Co., NY 1991, Chapters 8 and 11); Molecular Neurobiology, Z. Hall, editor (Sinauer Publ. Co., Sunderland, Mass. 1992, Chapters 11 and 12).
It is therefore an object of the present invention to provide a method for obtaining molecular signals that initiate regeneration of nerve connections in mammals.
It is a further object of the present invention to provide factors which initiate regeneration of nervous tissue in mammals.
It is another object of the present invention to provide methods for treatment of injuries to spinal cord and other central nervous system tissue.